Calculating temperature-dependent X-ray structure factors of α-quartz with an extensible Python 3 package.

The design of X-ray optics based on diffraction from crystals depends on the accurate calculation of the structure factors of their Bragg reflections over a wide range of temperatures. In general, the temperature dependence of the lattice parameters, the atomic positions and the atomic thermal vibrations is both anisotropic and nonlinear. Implemented here is a software package for precise and flexible calculation of structure factors for dynamical diffraction. α-Quartz is used as an example because it presents the challenges mentioned above and because it is being considered for use in high-resolution X-ray spectroscopy. The package is designed to be extended easily to other crystals by adding new material files, which are kept separate from the package's stable core. Python 3 was chosen as the language to allow the easy integration of this code into existing packages. The importance of a correct anisotropic treatment of the atomic thermal vibrations is demonstrated by comparison with an isotropic Debye model. Discrepancies between the two models can be as much as 5% for strong reflections and considerably larger (even to the level of 100%) for weak reflections. A script for finding Bragg reflections that backscatter X-rays of a given energy within a given temperature range is demonstrated. The package and example scripts are available on request. Also discussed, in detail, are the various conventions related to the proper description of chiral quartz.

Activation by TyrR in Escherichia coli K-12 by Interaction between TyrR and the α-Subunit of RNA Polymerase.

A novel selection was developed for mutants of the C-terminal domain of RpoA (α-CTD) altered in activation by the TyrR regulatory protein of Escherichia coli K-12. This allowed the identification of an aspartate to asparagine substitution at residue 250 (DN250) as an activation-defective (Act-) mutation. Amino acid residues known to be close to D250 were altered by in vitro mutagenesis, and the substitutions DR250, RE310, and RD310 were all shown to be defective in activation. None of these mutations caused defects in regulation of the upstream promoter (UP) element. The rpoA mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. The TyrR mutations EK139 and RG119 caused partial suppression of rpoA DN250, and TyrR RC119, RL119, RP119, RA77, and SG100 caused partial suppression of rpoA RE310. Additional activation-defective rpoA mutants (DT250, RS310, and EG288) were also isolated, using the chromosomal rpoA DN250 strain. Several new Act-tyrR mutants were isolated in an rpoA+ strain, adding positions R77, D97, K101, D118, R119, R121, and E141 to known residues S95 and D103 and defining the activation patch on the amino-terminal domain (NTD) of TyrR. These results support a model for activation of TyrR-regulated genes where the activation patch on the TyrR NTD interacts with the TyrR-specific patch on the α-CTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants as activator contact sites. IMPORTANCE Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene tyrR in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore, TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence and important plant interactions.

Biosynthesis of the Aromatic Amino Acids.

This chapter describes in detail the genes and proteins of Escherichia coli involved in the biosynthesis and transport of the three aromatic amino acids tyrosine, phenylalanine, and tryptophan. It provides a historical perspective on the elaboration of the various reactions of the common pathway converting erythrose-4-phosphate and phosphoenolpyruvate to chorismate and those of the three terminal pathways converting chorismate to phenylalanine, tyrosine, and tryptophan. The regulation of key reactions by feedback inhibition, attenuation, repression, and activation are also discussed. Two regulatory proteins, TrpR (108 amino acids) and TyrR (513 amino acids), play a major role in transcriptional regulation. The TrpR protein functions only as a dimer which, in the presence of tryptophan, represses the expression of trp operon plus four other genes (the TrpR regulon). The TyrR protein, which can function both as a dimer and as a hexamer, regulates the expression of nine genes constituting the TyrR regulon. TyrR can bind each of the three aromatic amino acids and ATP and under their influence can act as a repressor or activator of gene expression. The various domains of this protein involved in binding the aromatic amino acids and ATP, recognizing DNA binding sites, interacting with the alpha subunit of RNA polymerase, and changing from a monomer to a dimer or a hexamer are all described. There is also an analysis of the various strategies which allow TyrR in conjunction with particular amino acids to differentially affect the expression of individual genes of the TyrR regulon.

The TyrR regulon.

The TyrR protein of Escherichia coli can act both as a repressor and as an activator of transcription. It can interact with each of the three aromatic amino acids, with ATP and, under certain circumstances, with the C-terminal region of the alpha-subunit of RNA polymerase. TyrR protein is a dimer in solution but in the presence of tyrosine and ATP it self-associates to form a hexamer. Whereas TyrR dimers can, in the absence of any aromatic amino acids, bind to certain recognition sequences referred to as 'strong TyrR boxes', hexamers can bind to extended sequences including lower-affinity sites called 'weak TyrR boxes', some of which overlap the promoter. There is no single mechanism for repression, which in some cases involves exclusion of RNA polymerase from the promoter and in others, interference with the ability of bound RNA polymerase to form open complexes or to exit the promoter. When bound to a site upstream of certain promoters, TyrR protein in the presence of phenylalanine, tyrosine or tryptophan can interact with the alpha-subunit of RNA polymerase to activate transcription. In one unusual case, activation of a non-productive promoter is used to repress transcription from a promoter on the opposite strand. Regulation of individual transcription units within the regulon reflects their physiological function and is determined by the position and nature of the recognition sites (TyrR boxes) associated with each of the promoters. The intracellular levels of the various forms of the TyrR protein are also postulated to be of critical importance in determining regulatory outcomes. TyrR protein remains a paradigm for a regulator that is able to interact with multiple cofactors and exert a range of regulatory effects by forming different oligomers on DNA and making contact with other proteins. A recent analysis identifying putative TyrR boxes in the E. coli genome raises the possibility that the TyrR regulon may extend beyond the well-characterized transcription units described in this review.

Mode of action of the TyrR protein: repression and activation of the tyrP promoter of Escherichia coli.

The tyrP gene of Escherichia coli encodes a tyrosine specific transporter. Its synthesis is repressed by tyrosine but is activated by phenylalanine and to a lesser extent by tryptophan. Both of these effects are mediated by the TyrR protein when it binds to one or both of its cognate binding sites (TyrR boxes) which encompass nucleotides -30 to -75. Activation in the presence of phenylalanine or tryptophan involves a dimer binding to the upstream box and interacting with the alpha subunit (alphaCTD) of RNA polymerase (RNAP). Repression in the presence of tyrosine involves a hexamer binding to both TyrR boxes. The molecular basis for this repression has been studied in vitro. Whereas initial gel shift experiments fail to show the exclusion of RNAP from the promoter region when TyrR hexamer is bound, a DNase I analysis of slices from the gel shows that in the presence of TyrR, RNAP now binds to a previously unrecognized upstream promoter. Although this upstream promoter is bound strongly by RNAP and forms an open complex on linear DNA templates, it fails to form an open complex on supercoiled templates in vitro and is unable to initiate transcription in vivo. A subsequent gel shift assay using a tyrP fragment which eliminates the upstream RNAP binding site confirms conclusively that, in the presence of tyrosine and ATP, the TyrR protein prevents RNAP from binding to the tyrP promoter. In vitro studies have also been carried out in the presence of TyrR protein and phenylalanine. Binding of TyrR protein to the upstream TyrR box in the presence of phenylalanine is shown to increase the affinity of RNAP for the promoter and stimulate open complex formation at the -10 region of the tyrP promoter. This observation coupled with the results from mutational analysis supports the proposal that TyrR-phenylalanine activates tyrP transcription by stimulating the onset of open complex formation.

Molecular analysis of tyrosine-and phenylalanine-mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli.

The mechanism of repression of the tyrB promoter by TyrR protein has been studied in vivo and in vitro. In tyrR+ strains, transcription of tyrB is repressed by either tyrosine or phenylalanine. Both of the TyrR binding sites (strong and weak TyrR boxes) lie downstream of the tyrB transcription start site and are required for tyrosine- or phenylalanine-mediated repression. Our results establish that the binding of the TyrR protein to the weak box, induced by cofactor tyrosine or phenylalanine, is critical for repression to occur. Neither the binding of the TyrR protein dimer formed in the presence of phenylalanine, nor the binding of the hexamer formed in the presence of tyrosine, blocks the binding of RNA polymerase to the promoter. Instead, open complex formation is inhibited in the presence of tyrosine whereas a step(s) following open complex formation is inhibited in the presence of phenylalanine. Moving the TyrR boxes 3 bp or more further away from the promoter affects tyrosine-mediated repression without affecting phenylalanine-mediated repression which remains unaltered until 6 bp are inserted between the TyrR boxes and the promoter. Analysis of deletion and insertion mutants fails to reveal any face of the helix specificity for either tyrosine- or phenylalanine-mediated repression.